BACKGROUND: Cortisol measurement is useful in evaluation of Cushing syndrome, adrenal insufficiency, mineralocorticoid excess and congenital adrenal hyperplasia. We developed a liquid chromatography–tandem mass spectrometry (LCMS/MS) method for salivary and urinary cortisol and we determined the 95th percentile (p95) for the urinary and salivary cortisol. We compared them to the Mayo Clinic expected values. METHODS: Saliva at 8 am and 11 pm and 24h urine were obtained from 32 healthy (22 female, 34.3±9.3 yo) volunteers. We performed validation with the enoval software (Arlenda, Belgium). For the validation, we used water or urine with spiked known amounts of cortisol for the CORS and CTU respectively. For the CORS, samples were centrifuged, deuterium labelled cortisol was added as internal standard and the protein precipated by acetonitril. The supernatant was evaporated, dissolved in methanol acidified with acetic acid and analyzed by LCMS/MS. For CTU, samples were centrifuged, deuterium labelled cortisol was added as internal standard and diluted by the ammonium acetate and analyzed by LCMS/MS. At the Mayo Clinic, the expected values were 1-7.5 μg/L (7 a.m-9 a.m) and <1 μg/L (11-12 p.m) for CORS and 3.5-45 μg/24h (<18yo) for CTU. RESULTS: For the CTU, the with-in run did not exceed 3% (0.4-3%) and the between-run did not exceed 3.1% (0.4-3.1%) for 1.5-750 μg/L. The limit of quantification was 1.5 μg/L. The linearity was good between 1.5 and 750 μg/L. The recovery is 97.9±2.2% (95%CI for the mean: 92.4-101.1%). For the CORS, the with-in run and between run did not exceed 8% (1.9-8%) for 1.15-8.65 μg/L. The limit of quantification was 1.15 μg/L. The analyse presents a good linearity between 1.15 and 8.65 μg/L. The recovery is 99.9±2.9% (95%CI for the mean: 94.2-108.7%). The p95 for the CTU according to the CLSI C28-A3 was 33 μg/24h, and for the CORS was 5.42 μg/L at 8 am and 0.7 μg/L at 12 pm. CONCLUSIONS: Our developed method in liquid chromatography tandem mass spectrometry was validated for the measurement of urinary and salivary cortisol. Our findings indicate that the proposed analytical methods were suitable for routine purposes and useful in many pathological conditions.The expected values confirm these defined by the Mayo Clinic. [less ▲]

BACKGROUND: The enzym CYP24A1 catalyses the conversion of 25(OH)D3 in 24,25(OH)2D3. Recently, loss-of-function mutation of CYP24A1 has been identified in idiopathic infantile hypercalcemia (IIH). This ... [more ▼]

BACKGROUND: The enzym CYP24A1 catalyses the conversion of 25(OH)D3 in 24,25(OH)2D3. Recently, loss-of-function mutation of CYP24A1 has been identified in idiopathic infantile hypercalcemia (IIH). This genetic defect can be highlighted by high 1,25(OH)2D3 and undetectable 24,25(OH)2D3 levels. 24,25(OH)2D3 is also known to interfere with 25(OH)D3 determinations with immunoassays, leading to an overestimation of the 25(OH)D3 concentrations. We adapted the MassChrom kit on the AB SCIEX TQ 5500 in order to systematically provide, next to 25(OH)D3, 25(OH)D2 and C3 epimer, the concentrations of 24,25(OH)2D3. The aim of this study was to evaluate the performance of 24,25(OH)2D3 determination with this modified method. We also wanted to establish the reference value of 24,25(OH)2D3. METHODS: We modified the Chromsystems MassChrom method by adding the 24,25(OH)D3 correspondent transitions and performed a calibration by spiking known amounts of 24.25(OH)2D3. The LOQ was determined with 10 concentration levels of 24,25(OH)2D3. We selected 92 healthy children (40 girls; 2.4±1.51 years) presenting normal calcium levels (2.49±0.13mmol/l) to determine the 95th percentile (p95). RESULTS: The 24,25(OH)2D3 LOQ was 4.7 ng/ml. 85.9% of our subjects were below this LOQ. The p 95 for the 24,25(OH)2D3, according to the CLSI C28-A3, was <6.2 ng/ml. The average serum concentrations (mean±SD) of 25(OH)D3 and 3-epi-25(OH)D3 were 24.48±10.22ng/ml and 2.07±1.86 ng/ml respectively. The 24,25(OH)2D3 levels (r2=0.64) correlated with the 25(OH)D3 levels. CONCLUSIONS: Our adapted method from Chromsystems Vitamin D determination is available to quantify 24,25(OH)2D3. In this context, this method is able to determine high levels of 24,25(OH)2D3 that can possibly cross react with immunoassays. However, as the LOQ was not low enough, we couldn’t establish correct reference value for 24,25(OH)2D3. A derivatization step in the sample preparation would be interesting to improve the sensibility of the method. [less ▲]

Background: The aim of this work was to develop and validate a method for the determination of metanephrine (M), normetanephrine (NM) and methoxymetanephrine (METHO) in urine by liquid chromatography ... [more ▼]

Background: The aim of this work was to develop and validate a method for the determination of metanephrine (M), normetanephrine (NM) and methoxymetanephrine (METHO) in urine by liquid chromatography-tandem mass spectrometry (LCMS-MS) on the Triple Quad TQ 5500 from AB SCIEX. In fact, the determination of M and NM concentrations is used in clinical diagnosis of pheochromocytoma, a rare but potentially fatal tumor arising primarily from the chromaffin cells of the adrenal medulla. Methods: The samples were made of 24 hours acidified urines after centrifugation. Sample preparation was performed by hydrolysing and purifying by extraction column. After that, labeled M, NM and METHO were added as internal standard. Samples were analysed by liquid chromatography-electrospray tandem mass spectrometry. We determined the repeatability, reproducibility, accuracy profile and recovery on pooling urines samples from 9 volunteers analysed in triple run. Results: The results of the precision evaluation are shown in table. The repeatability did not exceed 8.4 % for M, 6.8% for NM and 10.8% for METHO. The concentration range was 71-781 µg/24h, 71-853 µg/24h and 20-854µg/24h for the M, NM and METHO respectively. The total precision did not exceed 12.5%, 11.8% and 8.8% for M, NM and METHO. The limit of quantification (LOQ) were 33.77µg/24h, 14.49µg/24H and 19.81 µg/24H for M, NM and METHO respectively. The accuracy varied from 99.69 to 100.2% for a range of 71 to 781 µg/24h, from 93.32 to 100.2% for a range of 71-853 µg/24h and from 99.85 to 100.6% for the range 20-854µg/24h for M, NM and METHO respectively. The recovery were 99.96% (95% CI for the mean: 96.5-103.4), 99.75% (96.5-102.9) and 100.08 (95.97-104.2) for the M, NM and METHO respectively. Conclusions: We have successfully developed and validated an LCMS-MS method to determine urinary M, NM and METHO on the TQ 5500 from AB SCIEX. It represents a convincing alternative to the HPLC method for a faster and reliable measurement of urinary M, NM and METHO. [less ▲]

Background: Twenty-five hydroxy-vitamin D (25(OH) D) determination is now routinely prescribed in the Laboratory. Recently, different new methods have been available for this determination. Among them ... [more ▼]

Background: Twenty-five hydroxy-vitamin D (25(OH) D) determination is now routinely prescribed in the Laboratory. Recently, different new methods have been available for this determination. Among them, LCMS/MS methods have emerged in some laboratories. However these methods are generally “home-brewed” and an important variability between them can be seen on different external quality controls, mainly due to a lack of standardization. Recently, Perkin-Elmer (PE) (Turku, Finland) and Chromsystem (CS) (Grafelfing, Germany) launched a standardised method for 25(OH )D determination on LCMS/MS. The aim of our study was to compare these methods on the AB SCIEX TQ5500 (Framingham, Massachusetts, USA) LCMS/MS to measure 25(OH) D3. Methods All the samples were treated according to our preanalyitical procedure: after sampling, they were spun at +4°c at 3500G, aliquoted and kept frozen at -20°c until determination. A method comparison was assessed with CS and PE for the measurement of the 25(OH)D3. We selected 110 remnant samples with 25(OH)D3 levels ranging from 1.6 to 136.7 ng/ml with the PE method to cover the range of usually values Slope and intercept were calculated using Passing and Bablock linear regression and we compared the methods with the Bland and Altman plots. Results For CS, the method is linear up to 250 µg/L, the LOQ is 3.6 µg/L, the intra-assay CV is < 5% and the inter-assay is < 7%. For PE, the method is linear up to 314 µg/L, the LOQ is 3.4 µg/L, the intra-assay CV is < 7.8% and the inter-assay is < 8.5%. On the whole range of measure (n=110), the regression equation is PE = 0.8521+0.9226 (CS) (95%CI of the intercept: (-0.0048;1.37) and 95% CI of the slope (0.89;0.95). The Bland and Altman plot does not show any bias between the two methods (mean difference CS-PE= -2.5 ng/ml) and the standard deviation of the mean is 3,98 ng/ml Conclusion: The performances of these methods are comparable on our new TQ 5500 from AB SCIEX. For now, there is no consensus on a “reference” method for vitamin D quantification. We notice only that the values obtained by CS are systematically a little bit lower than PE’s values, especially for results below 20 ng/ml. However, we have no clear explanation for such behaviour. [less ▲]